Pharmaceutical composition and process for isolation of trans-tetracos-15-enoic acid and method of treatment for hepatotoxicity

ABSTRACT

The present invention relates to a pharmaceutical composition comprising a bioactive constituent trans-tetracos-15-enoic acid and a process for the isolation of tetracos-15-enoic acid from the plant  Indigofera tinctoria  along with a method of treatment for hepatotoxicity.

FIELD OF INVENTION

[0001] The present invention relates to a pharmaceutical compositioncomprising trans-tetracos-enoic acid and a process for the isolation oftrans-tetracos-15-enoic acid from a plant source Indigofera tinctoria.The present invention also relates to a method of treatment forhepatotoxicity.

BACKGROUND AND PRIOR ART REFERENCES

[0002]Indigofera tinctoria Linn. (Family Leguminosae, Hindi-Neel) is anannual herbaceous shrub, 4-6 ft. high, found throughout India. Earlierit was cultivated in India, China and other eastern countries as asource of Indigo.

[0003] In an indigenous system of medicine, extract of this plant isused for the treatment of epilepsy, nervous disorders and bronchitis[Wealth of India, vol.5 (Council of Scientific and Industrial Research,New Delhi) 182, (1959)]. The plant is also known to be used as anointment for sores, old ulcers and haemorrhoids [R.N. Chopra, S. L.Nayar and I C Chopra, Glossary of Indian Medicinal Plants, 141 (1956)].The leaves of the plant have been used in liver ailments [Nadkarni, K.M., Indian Materia Medica, vol.1 (Popular Book Depot, Bombay, 680(1954)].

[0004] Organic solvent extract of the leaves of the plant exhibitedmarked hepatoprotective effect against carbon tetrachloride inducedhepatic injury in rabbits, rats and mice [Anand, K. K., Chand Dewan,Ghatak, B. J. Ray & Arya, R. K., Indian J. Expl. Biol. 19, 298 (1981),Anand, K. K., Chand Dewan & Ghatak, B. J. Ray, Indian J. Expl. Biol.,17, 685(1979)].

[0005] Literature survey revealed that earlier reports showed thepresence of trans-tetracos-15-enoic acid in Jojoba oil ex. Simmondsiachinensis seeds (0.62-1.11%), c/s isomer of the acid in fatty acids ofthe seed oil of. Microula sikkimensis (1.2%). [Wang, Huiying, Yu,Xuefian, Yi, Yuanfen & Ding, Jingkai, Yunnan Zhiwu Yanjiu 1989 11 (I),60-4 (Ch.), L, Jing Jingmin; Wang, Jingping; Yu, Feuglau. Zhiwn, Xuebao,1989, 31 (1) 50-3; (Ch). These reports do not mention isolation of theconstituent and the content estimation is based on GLC data.

[0006] The bioactive constituent thus isolated in the present inventionfrom the plant Indigofera tinctoria is designated as RLJ-NE-598(025)(F)(A₃) (TCA)

[0007] Hepatotoxins that cause acute hepatitis have close resemblancewith the viral hepatitis-clinically, biochemically and histologically.Drugs also cause of chronic hepatic disease as chronic hepatitis, fattyliver, cirrhosis and several vascular lesions of the liver.

[0008] In many instances drug induced hepatitis proves indistinguishablefrom viral hepatitis. Chemically induced hepatic injury for experimentalstudies should be severe enough to cause cell death or to modify hepaticfunctions. The mechanism of acute hepatic injury depends upon thechemical compound and the species of animals used. Many chemicalsproduce parenchymal damage, arrest bile flow and cause jaundice(choleretic injury).

[0009] In the present invention the applicants have studiedhepatoprotective activity against CCl₄, paracetamol (APAP,acetaminophen), D-galactosamine and alcohol induced hepatotoxicity.

OBJECTS OF THE INVENTION

[0010] The main object of the present invention is to provide apharmaceutical composition comprising a bioactive compoundtrans-tetracos-15-enoic acid.

[0011] Another object of the present invention is to provide a processfor the isolation of trans-tetracos-15-enoic acid from a plant sourceIndigofera tinctoria having significant dose-dependent hepatoprotectiveactivity.

[0012] Another object of the present invention is to provide a wellidentified single constituent of plant origin for use in therapeutics.

[0013] Yet another object of the present invention provides a method oftreatment of mammals and humans for hepatotoxicity.

SUMMARY OF THE INVENTION

[0014] The present invention relates to a pharmaceutical compositioncomprising a bioactive compound trans-tetracos-15-enoic acid and aprocess for the isolation of trans-tetracos-15-enoic acid from a plantsource viz., Indigofera tinctoria. The present invention also provides amethod for the treatment of acute hepatitis using this compound.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Accordingly, the present invention provides a pharmaceuticalcomposition comprising an effective amount of bioactivetrans-tetracos-15-enoic acid extracted from a plant source Indigoferatinctoria, used to treat subjects with hepatic disorders.

[0016] An embodiment of the present invention, wherein said compositionalong with dehydrocholic acid is used for the enhancement of cholreticactivity.

[0017] Another embodiment of the present invention whereintrans-tetracos-15-enoic acid is used singly or in combination withpharmaceutically acceptable additives.

[0018] Yet another embodiment of the present invention wherein thepharmaceutically acceptable additives are selected from the groupconsisting of carriers, diluents, solvents, filters lubricants,excipients, binder or stabilizers.

[0019] Still another embodiment of the present invention wherein thesaid composition is used for both preventive and curative properties.

[0020] Yet another embodiment of the present invention wherein saidcomposition is used systemically, orally or by any clinically/medicallyaccepted methods.

[0021] Still another embodiment of the present invention wherein thecomposition is used to treat hepatic disorders that are clinically,biochemically and histologically similar to that of viral hepatitis,chronic hepatitis, fatty liver, cirrhosis and several vascular lesionsof the liver.

[0022] Yet another embodiment of the present invention wherein saidcomposition is used to treat damage induced by hepatotoxins.

[0023] Still another embodiment of the present invention wherein thehepatotoxins are selected from the group comprising Galactosamine,Paracetamol, Carbon tetrachloride and alcohol.

[0024] Yet another embodiment of the present invention wherein thesubjects is selected from the group consisting of mammals and humans,preferably humans.

[0025] Still another embodiment of the present invention wherein thedosage for treating CCl₄ induced hepatotoxicity in mammals is in therange of 10-100 mg/kg body weight.

[0026] Yet another embodiment of the present invention wherein thepreferred dosage for treating CCl₄ induced hepatotoxicity in mammals isin the range of 50-100 mg/kg body weight.

[0027] Still another embodiment of the present invention wherein thehepatoprotective activity in CCl₄ induced hepatotoxic mammals is upto88%.

[0028] Yet another embodiment of the present invention wherein thedosage for treating acetaminophen induced hepatotoxicity in mammals isin the range of 10-100 mg/kg body weight.

[0029] Still another embodiment of the present invention wherein thepreferred dosage for treating acetaminophen induced hepatotoxicity inmammals is in the range of 50-100 mg/kg of body weight.

[0030] Yet another embodiment of the present invention wherein thehepatoprotective activity in acetaminophen induced hepatotoxicity inmammals is upto 86%.

[0031] Still another embodiment of the present invention wherein thedosage for treating Galactosamine induced hepatotoxicity in mammals isin the range of 10-50 mg/kg of body weight.

[0032] Yet another embodiment of the present invention wherein thepreferred dosage for treating D-Galactosamine induced hepatotoxicity isin the range of 25-50 mg/kg of body weight.

[0033] Still another embodiment of the present invention wherein thehepatoprotective activity in Galactosamine induced hepatotoxicity inmammals is upto 73%.

[0034] Yet another embodiment of the present invention wherein thedosage for treating alcohol induced hepatotoxicity in mammals is in therange of 10-50 mg/kg of body weight.

[0035] Still another embodiment of the present invention wherein thepreferred dosage for treating alcohol induced hepatotoxicity is in therange of 25-50 mg/kg of body weight.

[0036] Yet another embodiment of the present invention wherein thehepatoprotective activity in alcohol induced hepatotoxicity in mammalsis upto 100%.

[0037] Still another embodiment of the present invention wherein thepreferred dosage for treating hepatic disorders in mammals is 1 about50-100 mg/kg of body weight.

[0038] Yet another embodiment of the present invention wherein thepreferred dosage for treating hepatic disorders in human beings is about10-105 mg/kg of body weight.

[0039] The invention also provides a process for the isolation oftrans-tetracos-15-enoic acid, a bioactive constituent from plantIndigofera tinctoria, said process comprising the steps of:

[0040] (a) extracting the powdered plant parts with an aliphatichydrocarbon solvent at a temperature in the range of 60-80° C. for about20-30 hours;

[0041] (b) triturating the extract of step (a) with a ketonic solvent;

[0042] (c) separating the solvent soluble portion of step (b) and theresidue by using silica gel bed;

[0043] (d) subjecting the residue of step (c) to column chromatographyby eluting with a mixture of organic solvents of increasing polarity toyield a bioactive fraction; and

[0044] (e) resolving fraction as obtained in step (d) by highperformance liquid chromatography and crystallizing with an alcoholicsolvent to obtain the bio-active constituent trans-tetracos-15-enoicacid.

[0045] Yet another embodiment of the present invention whereinalternatively, the fraction obtained in step (d) when subjected tosemipreparative high performance liquid chromatography, yields bioactiveconstituent trans-tetracos-15-enoic acid.

[0046] Still another embodiment of the present invention wherein theplant parts used are aerial parts selected from the group consisting ofleaves, stem and bark.

[0047] Yet another embodiment of the present invention wherein thehydrocarbon solvent is selected from the group consisting of petroleumethane, n-hexane, cyclohexane or ligroin and preferably petroleum ether.

[0048] Still another embodiment of the present invention wherein in theketonic solvent is selected from the group consisting of acetone,ethyl-methyl-ketone, or methyl isobutyl ketone.

[0049] Yet another embodiment of the present invention wherein thesolvents used for eluting the column to obtain hepatoprotective fractionare selected from the group consisting of petroleum ether, n-hexane,chloroform, ethyl acetate, a mixture thereof and preferably a mixture ofpetroleum ether and ethyl acetate.

[0050] Still another embodiment of the present invention wherein in step(d), the solvent used for performing HPLC is a mixture of Acetonitrileand H₂O in the ratio of 95:5.

[0051] Yet another embodiment of the present invention wherein in step(e) the HPLC purified compound is further purified by crystallizing withan alcoholic solvent selected from methanol and isopropanol.

[0052] Still another embodiment of the present invention wherein thehepatoprotective fraction thus obtained is designated as Indigotoneconsisting of a mixture of three compounds.

[0053] Yet another embodiment of the present invention wherein thepercentage of mixture of three compounds of hepatoprotective fraction is11-12%, 12-13% and 75-77% respectively.

[0054] The present invention further provides a method of treatingsubjects with liver disorders, the said method comprising administeringa pharmaceutically effective dosage of trans-tetracos-15-enoic acid.

[0055] Yet another embodiment of the present invention, a method whereintrans-tetracos-15-enoic acid is used to treat liver disorders caused byGalactosamine, Paracetamol, Carbon tetrachloride and alcohol.

[0056] Still another embodiment of the present invention, a methodwherein the dosage for CCl₄ induced hepatotoxicity in mammals is in therange of 10-100 mg/kg body weight.

[0057] Yet another embodiment of the present invention, a method whereinthe preferred dosage for CCl₄ induced hepatotoxicity in mammals is inthe range of 50-100 mg/kg body weight.

[0058] Still another embodiment of the present invention, a methodwherein the hepatoprotective activity in CCl₄ induced hepatotoxicmammals is upto 88%.

[0059] Yet another embodiment of the present invention, a method whereinthe dosage for acetaminophen induced hepatotoxicity in mammals is in therange of 10-100 mg/kg body weight.

[0060] Still another embodiment of the present invention, a methodwherein the preferred dosage for acetaminophen induced hepatotoxicity inmammals is in the range of 50-100 mg/kg of body weight.

[0061] Yet another embodiment of the present invention, a method whereinthe hepatoprotective activity in acetaminophen induced hepatotoxicity inmammals is upto 86%.

[0062] Still another embodiment of the present invention, a methodwherein the dosage for Galactosamine induced hepatotoxicity in mammalsis in the range of 10-50 mg/kg of body weight.

[0063] Yet another embodiment of the present invention, a method whereinthe preferred dosage for D-Galactosamine induced hepatotoxicity is inthe range of 25-50 mg/kg of body weight.

[0064] Still another embodiment of the present invention, a methodwherein the hepatoprotective activity in Galactosamine inducedhepatotoxicity in mammals is upto 73%.

[0065] Yet another embodiment of the present invention, a method whereinthe dosage for alcohol induced hepatotoxicity in mammals is in the rangeof 10-50 mg/kg of body weight.

[0066] Still another embodiment of the present invention, a methodwherein the preferred dosage for alcohol induced hepatotoxicity is inthe range of 25-50 mg/kg of body weight.

[0067] Yet another embodiment of the present invention, a method whereinthe hepatoprotective activity in alcohol induced hepatotoxicity inmammals is upto 100%.

[0068] Still another embodiment of the present invention, wherein thedosage for the enhancement of cholretic activity is in the range of20-50 mg/kg of body weight.

[0069] Yet another embodiment of the present invention, the enhancedcholretic activity in mammals is upto 43%.

[0070] Still another embodiment of the present invention, a methodwherein trans-tetracos-15-enoic acid is used singly or in combinationwith pharmaceutically acceptable carriers.

[0071] Yet another embodiment of the present invention, a method whereinthe trans-tetracos-15-enoic acid is administered to a subject incombination with pharmaceutically acceptable additives, carriers,diluents, solvents, filters, lubricants, excipients, binder orstabilizers.

[0072] Still another embodiment of the present invention, a methodwherein the desired dosage is administered for both preventive andcurative properties.

[0073] Yet another embodiment of the present invention, a method whereintrans-tetracos-15-enoic acid is administered systemically, orally or byany clinically/medically accepted methods.

[0074] Still another embodiment of the present invention, a methodwherein the subject is selected from animals, mammals, and preferablyhumans.

[0075] Still yet another embodiment of the present invention, a methodwherein the preferred dosage for hepatic disorders in mammals is 1 about50-100 mg/kg of body weight.

[0076] Yet another embodiment of the present invention, a method whereinthe preferred dosage for human beings is about 10-15 mg/kg of bodyweight.

[0077] The invention is further explained in the form of followingpreferred embodiments.

[0078] Chromatographic fractionation of Indigofera tinctoria extract bya host of procedures viz., CC, MPLC & Centrifugal partitionchromatography yielded four fractions designated as A, B, C & Drespectively.

[0079] Pharmacological evaluation of these fractions showed that thehepatoprotective activity has got concentrated in fraction D, whichexhibited dose-dependant response from 6.25 mg-Kg−1 P.O. in experimentalanimals. HPLC NP-SiO2, 10 mμ CH₃OH:CHCl₃ 10:90, or RP-C18, CH₃CN:H₂O95:5, isocratic elution, λ=254 nm) indicated fraction D to be composedof three constituents, compound A-11-12%, Compound B=12-13% and compoundC=75-77%.

[0080] The fraction, which is designated as ‘Indigotone’ on CC and semipreparative HPLC yielded pure compound C. The compound onpharmacological evaluation showed dose dependent hepatoprotectiveactivity in experimental animals from 12.5 mg-Kg−1 onwards.

[0081] Compound (C) is determined to be trans-tetracos-15-enoic acid onthe bases of spectral data, viz., IR, UV, NMR (1H & 13C) & MS data.

[0082] This constitutes first observation and isolation oftrans-tetracos-15-enoic acid from Indigofera tinctoria.

[0083] The hepatoprotective constituent obtained by the process of thepresent invention is present to the extent of 0.09-0.11% on the basis ofdried plant material.

[0084] The invention is described with reference to the examples givenbelow which should not, however, be construed to limit the scope ofpresent invention.

[0085] Treatment with Bio-Active Compound of the Present Invention andSilymarin (Conventional Drug):

[0086] Freshly prepared suspension (1%, w/v) in 0.2% gum acacia innormal saline is used for all the experiments except for toxicitystudies where (10%, w/v) suspension is used. Silymarin suspension (1%,w/v) in 0.2% gum acacia is used as a reference standard (positivecontrol).

EXAMPLE I

[0087] Dried aerial portion of the plant (1.3 Kg) is powdered,Soxhletted with petroleum ether (60-80° C.) for 30 hours. The resultantextract is concentrated and dried. DarkGreen semisolid residue (31 g),is triturated with acetone (5×200 ml) and acetone soluble portion isconcentrated under reduced pressure to get a residue (21 g). The residueis adsorbed on SiO₂ gel (63 g, 100-200 mesh) and charged on a glasscolumn (diameter 5-cm, height 85 cm), packed with SiO2 gel (250 g,100-200 mesh). The column is eluted with petroleum ether: ethyl acetatemixtures of increasing polarity and finally with ethyl acetate.

[0088] The fractions eluted with petroleum ether: ethyl acetates (95:5)are pooled and the residue is designated as ‘Indigotone’, a mixture ofthree compounds (A-12%, B-14%, and C-74%). The fraction is subjected toHPLC (RP-18, 10 mμ, CH₃CN:H₂O, 95:5, λmax 254) to yield 1.1 g of pure C.Further purification is attained by crystallization from methanol (−20°C.). Purified constituent (m.p. 61° C.) is determined to betrans-tetracos-15-enoic acid on the base of co-TLC and spectral data.

EXAMPLE 2

[0089] Dried and powdered aerial portion of the plant material (650 g)is extracted with boiling hexane (2.5L) for 20 hours. The resultingextract is subjected to desolventation under reduced pressure yielding adark green viscous residue (16 g). The residue is triturated with ethylmethyl ketone (4×250 ml) and extracted material is concentrated anddried to get (11 g) of a semisolid material. The material is observed tobe a complex mixture on TLC plate and thus is subjected to CC on SiO₂gel. The column is eluted with (a) dichloromethane (1.5L), (b)dichloromethane-ethyl acetate (98:2,2L), (c) dichloromethane-ethylacetate (95:5, 2L), (d) dichloromethane-ethyl acetate (1L). Solidresidue (1 g) obtained from fraction (d) is designated as ‘Indigotone’.HPLC output indicated Indigotone to be composed of three constituentsviz., the most polar compound (A) 12%, constituent of medium polarity(B) 13% and the least polar compound (C) 75%.

[0090] Semipreparative HPLC (RP-C18, 10 mμ, CH₃CN:H₂O 95:5, λmax 254 nm)with isocratic elution yielded pure C (600 mg). It is further purifiedby crystallization from isopropanol (−20° C.). Purified constituents,m.p. (61° C.) is identified to be trans-tetracos-15-enoic acid on thebasis of spectral data, trans-tetracos-15-enoic acid has been evaluatedfor its hepatoprotective activity against CCl₄ hepatotoxin usingcommercially available Silymarin as reference materials. The comparativedata have been provided in the TABLE-1.

[0091] To assess the efficacy of the drug, pharmacological studies areconducted on Wistar albino rats (150-180 g) and Swiss albino mice (25-30g) of either sex, colony—bred in-house. After procurement, all theanimals are divided into different groups and are left for one week foracclimatization in an experimentation room and are maintained onstandard conditions (23±2° C., 60-70% relative humidity and 12 hoursphoto period). The animals are fed with standard rodents' pellet dietand water ad libitum. There are six animals in each group except forgeneral behavior and acute toxicity studies where ten animals are usedin each group.

[0092] The following hepatotoxins are used in the present invention toassess the efficacy of the drug:

[0093] (a) Carbon tetrachloride (CCl₄): One of the most powerfulhepatotoxins (in terms of severity of injury) Carbon tetrachloride(CCl₄) as it causes toxic necrosis which leads to biochemical changeshaving clinical features similar to those of acute viral hepatitis(Vogel, 1977, Bramanti et. al., 1978, Kumar et. al., 1992). Liver injuryis produced by administration of CCl₄ mixed with liquid paraffin.Animals are given single dose of CCl₄ (50 μl.kg⁻¹, p.o.) in acute singletreatment and (0.5 ml.kg⁻¹, p.o.) in case of multi-treatment with drug.It is administered orally (p.o) by gastric intubation. The controlanimals received the equal volume of liquid paraffin. The results havebeen recorded in TABLES 1-5

[0094] (b) Paracetamol (APAP, acetaminophen): It is a therapeutic agentwidely used as analgesic/antipyretic drug. When taken in large doses itcauses hepatic necrosis which leads to biochemical changes havingclinical features similar to those of acute viral hepatitis in humans(Proudfoot and Wright, 1970). The similar effect is observed in animals.The toxic effect can be potentiated if it is given several hours afterthe anesthetic ether inhalation (Wells et. al., 1985). Liver injury isinduced by injecting paracetamol (200 mg.kg⁻¹) interaperitoneally innormal saline (pH 9.4) six hour after inhalation of anesthetic ether (4ml/4 min/6 animals) in a closed chamber. The control animals receivedthe equal volume of vehicle. The results have been recorded in TABLES6-7.

[0095] (c) D-Galactosamine: It is one of the toxins that induce hepaticinflammatory conditions in the rat liver that clinically resembles toviral hepatitis. The mechanism of GalN induced liver injury has beenextensively examined and this model is now accepted as one of theauthentic systems of liver damage (Bauer et. al., 1974, Al-Tuwaijiri et.al., 1981).

[0096]  The data have been recorded in TABLES 8-9.

[0097] Hepatic damage is produced by injecting GalN (300 mg.kg⁻¹)subcutaneously in normal saline. The control animals received the equalvolume of vehicle.

EXAMPLE 3

[0098] Hexobarbitone Induced Narcosis:

[0099] The different doses of TCA, silymarin and vehicle (normal saline)are fed to different groups of mice only once at 1 hour beforehepatotoxin (CCl₄, 50 μl.kg⁻¹, p.o.) administration. Animals are givenHexobarbitone (60 mg.kg⁻¹, i.p.) 2 hours after hepatotoxin and “sleeptime” (Fujimoto, 1960; Dreyfuss, 1970) of all the animals is monitored.The time of onset of loss of righting reflex to the recovery in minutesis taken as the duration of sleep. (Table 1).

EXAMPLE 4

[0100] Zoxazolamine-Induced Paralysis:

[0101] The different doses of TCA, silymarin and vehicle (normal saline)are fed to different groups of mice only once 1 hour before hepatotoxin(CCl₄, 50 μl.kg⁻¹, p.o.) administration. Animals are given Zoxazolamine(70 mg.kg⁻¹, i.p.) 2 hours after hepatotoxin and “paralysis time”(Conney, 1960) of all the animals is monitored. The time of onset ofparalysis to the recovery in minutes is taken as the duration ofparalysis. (Table 1).

EXAMPLE 5

[0102] Effect on Serum and Hepatic Biochemical Parameters:

[0103] I) CCl₄ induced hepatotoxicity:

[0104] (a) Treatment of test material before and after hepatotoxin: Thedifferent doses of, silymarin and vehicle (normal saline) are fed todifferent groups of rats at 48, 24, 2 hours before and 6 hours afterhepatotoxin (CCl₄, 0.5 ml.kg⁻¹, p.o.) intoxication. Blood is collectedfrom orbital sinus in all the animals 18 hours after last treatment andserum separated for different estimations. All the animals are thenkilled by decapitation, their livers are quickly excised, cleaned ofadhering tissue, weighed and homogenized in phosphate buffer saline forthe analysis of hepatic parameters (Agarwal and Mehendale, 1983,Klingensmith and Mehendale, 1982, Zimmerman, 1973, Edmondson and Peter,1985, Mitchell, et al, 1973). (Tables 2-4).

[0105] (b) Treatment of Test Material After Hepatotoxin (Curative Study,Post Treatment)

[0106] The different doses of TCA, silymarin and vehicle (normal saline)are fed to different groups of rats at 6, 24 and 48 hours, afterhepatotoxin (CCl₄, 0.5 ml.kg⁻¹, p.o.) intoxication. Blood is collectedfrom the orbital sinus in all the animals 2 hours after last treatmentand serum separated for different estimations. A portion of the liver isprocessed for histopathological studies. (Table 5).

[0107] II) Paracetamol Induced Hepatotoxicity:

[0108] (a) Treatment of Test Material Before and After Hepatotoxin:

[0109] The different doses of TCA, silymarin and vehicle (normal saline)are fed to different groups of mice at 72, 48, 24 hours, 1 hour beforediethyl-ether inhalation and 1 hour after hepatotoxin (paracetamol, 200mg.kg⁻¹, i.p.) given 6 h after exposure to diethyl-ether. Blood iscollected from orbital sinus in all the animals 18 hours after lasttreatment and serum separated for different estimations. A portion ofthe liver is processed for histopathological studies. (Table 6)

[0110] (b) Treatment of Test Material After Hepatotoxin (Curative Study,Post Treatment)

[0111] The different doses of TCA, silymarin and vehicle (normal saline)are fed to different groups of mice at 1, 24, 48 hours, and 72 hoursafter hepatotoxin (paracetamol, 200 mg.kg⁻¹, i.p.). intoxication. Bloodis collected from the orbital sinus in all the animals 2 hours afterlast treatment and serum separated for different estimations. All theanimals are then killed by decapitation, their livers are quicklyexcised, cleaned of adhering tissue, weighed and homogenised inphosphate buffer saline for the analysis of hepatic parameters. Aportion of the liver is processed for histopathological studies. (TABLE7)

[0112] III) D-Galactosamine Induced Hepatotoxicity:

[0113] (a) Treatment of Test Material Before and After Hepatotoxin:

[0114] The different doses of TCA, silymarin and vehicle (normal saline)are fed to different groups of mice at 48, 24, 2 hours before and 6hours after hepatotoxin (GalN, 300 mg.kg⁻¹, s.c.) intoxication. Blood iscollected from orbital sinus in all the animals 18 hours after lasttreatment and serum separated for different estimations. All the animalsare then killed by decapitation, their livers are quickly excised,cleaned of adhering tissue, weighed and homogenised in phosphate buffersaline for the analysis of hepatic parameters. A portion of the liver isprocessed for histopathological studies. (Table 8)

[0115] (b) Treatment of Test Material After Hepatotoxin (Curative Study,Post Treatment)

[0116] The different doses of, silymarin and vehicle (normal saline) arefed to different groups of rats at 6, 24, and 48 hours, afterhepatotoxin (GalN, 300 mg.kg⁻¹, s.c.) intoxication. Blood is collectedfrom the orbital sinus in all the animals 2 h after last treatment andserum separated for different estimations. All the animals are thenkilled by decapitation, their livers are quickly excised, cleaned ofadhering tissue, weighed and homogenised in phosphate buffer saline forthe analysis of hepatic parameters. A portion of the liver is processedfor histopathological studies. (Table 9)

[0117] IV) Alcohol Induced Hepatotoxicity:

[0118] Liver lesions are induced by daily feeding of alcohol 376 mg.kg⁻¹(p.o.) for six weeks to the different groups of rats. In the last twoweeks of feeding of alcohol, different doses of TCA or silymarin arealso fed daily 30 minutes after alcohol to the respective groups. Bloodis collected from the orbital sinus in all the animals 2 h after thelast treatment administered and serum separated for differentestimations. All the animals are then killed by decapitation, theirlivers are quickly excised, cleaned of adhering tissue, weighed andhomogenised in phosphate buffer saline for the analysis of hepaticparameters. (Tables 10&11)

[0119] Parameters Studied:

[0120] GPT and GOT: Pyruvate formed by transamination reaction isdetermined spectrophotometerically after reaction with2,4-dinitrophenylhydrazine (Reitman and Frankel, 1957).

[0121] ALP: p-nitrophenol formed in alkaline medium is measuredspectrophotometerically using p-nitrophenyl phosphate as substrate(Waler and Schutt, 1974).

[0122] Bilirubin: Total bilirubin is measured by diazotization reactionwith NaNO₂ (Malloy and Evelyn, 1937)

[0123] Triglycerides: Triglycerides from serum are extracted withisopropanol and sopanified with KOH. The liberated glycerol is convertedto formaldehyde by periodate and determined after reaction with acetylacetone. Triolein is used as standard (Neri and Firings, 1973).

[0124] Glutathione: It is determined after de-proteination by reactionwith DTNB (Ellman 1959 as modified by David 1987).

[0125] Lipid peroxidation: Thiobarbituric acid reacting substances aredetermined spectrophotometerically at 535 nm.

[0126] Drug metabolising enzymes: N-demethylase and Aniline hydroxylaseare determined by spectrophotometeric methods however NADPH is usedinstead of NADPH generating system. (Imai Y, et al, 1966)

[0127] G-6-pase: Inorganic phosphate liberated from glucose-6-phosphateis measured with Amminonapthol sulphonic acid after reaction withammonium molybdate. (Huijing, F., 1974) Glycogen and enzymes of glycogenmetabolism. In: Clinical Biochemistry, Principles and Methods., H. Ch.Curtius and M. Roth (Eds.) Vol. 2., Walter de. Gruyter, Berlin., N.Y.Pp. 1208-1235.

[0128] SDH: Succinate dehydrogenase activity is measured using potassiumferricyanide in phosphate buffered medium. Slater and Bonner. (1952).Effect of fluoride on the Succinate oxidase system. Biochemical Journal52, 185-196.

[0129] Protein: The protein is precipitated with TCA and dissolved inalkaline medium. The protein is measured by Folin's reagent. Lowery, O.H., Rosebrough, N. J., Farr, A. L. and Randle, R. J. (1951). Proteinmeasurement with the Folin phenol Reagent. J. Biol. Chem. 193, 265-275.

[0130] GGT: p-nitroaniline formed γ-glutamyl p-nitroanilide inglycylglycine buffer measured at 405. nm. Szasz, g. (1976). Clin. Chem.22, 2051-2055.

[0131] Hepatoprotective Activity:

[0132] Hepatoprotective activity (H) is calculated by the followingequation:

H=[1−(TC−V/VC−V)]×100

[0133] Where TC, VC, and V are drug+toxin, vehicle+toxin and vehicletreated groups of animals respectively.

EXAMPLE 6

[0134] Effect on Bile Flow and Bile Solids

[0135] The liver, by producing bile, plays an important role indigestion. The presence of bile in the intestine is necessary toaccomplish the digestion and absorption of fats as well as absorption ofthe fat-soluble vitamins—A, D, E & K. Bile is also an important vehicleof excretion. It removes many drugs, toxins, bile pigments and variousinorganic substances either derived from the diet or synthesized by thebody as cholesterol or as cholic acid. Increase in the bile flow issuggestive of stimulating action of liver microsomal enzymes. Effect onthe liver bile flow of test drug and that of vehicle is carried outafter cannulating the bile duct in normal anaesthesied rats. Bilecollected is from each animal from 0-5 hours (Klaassen, 1969, Donal etal. 1953). (TABLE 12)

[0136] Histopathological Studies:

[0137] A portion of the liver after treatment of hepatotoxin (GalN,CCl_(4,) and paracetamol) and test material is processed forhistopathological studies by routine hematoxyline and eosin stainedsections (Krajian, A. A., 1963).

[0138] General Behavior and Acute Toxicity:

[0139] Using different doses (10, 30, 100, 1200, 1400, 1600, 1800 and2000 mg.kg⁻¹) of TCA given orally to the groups of 10 mice for eachdose, while one group with same number of mice served as control. Theanimals are observed continuously for 1 hour and then half hourly for 4hours for any gross behavioral changes and general motor activity,writhing, convulsion, response to tail pinching, gnawing, piloerection,pupil size, fecal output, feeding behaviour etc. and further up to 72hours for any mortality. Acute LD₅₀ values in mice are calculated by themethod of Miller and Tainter, (1944). Mortality of animals in all thegroups used in different models for determining hepatoprotectiveactivity during the period of treatment is also recorded as a roughindex of subacute toxicity.

[0140] Statistical analysis: The data obtained are subjected tostatistical analysis using ANOVA for comparing different groups(Armitage, 1987) and Dunnett's t test for control and test groups(Dunnett, 1964). The regression coefficient (Slope b) correlationcoefficient (r) with its p value and ED₅₀ with 95% confidence limit (CL)are determined by regression analysis using log dose and percent effectof adaptogenic activity (Swinscow, 1980). The two tailed paired studentt test for comparing means before and after treatment and one tailedunpaired student t test for comparing control and drug treated groups(Ghosh, 1984) are used. The p value of less than 0.05 or less is takenas the criterion of significance.

[0141] These results clearly show that TCA obtained from the plantIndigofera tinctoria when given orally exhibited dose dependenthepatoprotective activity in pre-and post-treatment against CCl₄,paracetamol and galactosamine induced acute hepatic injury.

[0142] Human Dose:

[0143] Doses for human being can be calculated by equivalent surfacearea doses conversion factor (equivalency on the basis of mg/sqm).

ADVANTAGES OF THE PRESENT INVENTION

[0144] 1. Commercially available herbal hepatoprotective are notstandardized chemically as well as biologically. Since the bioactivityof herbal preparations is mainly ascribed to secondary metabolites,efficacy is not achieved unless such preparations are standardized onthe basis of a few chemical constituents.

[0145] 2. Latest hepatoprotective drug marketed by Ranbaxy (India) Ltd.viz., Silymarin is a mixture of three constituents, whose relativeproportions also varies from batch to batch.

[0146] 3. Present invention provides a single bioactive constituent fromIndigofera tinctoria having dose dependent activity in experimentalanimals against CCl₄, paracetamol and galactosamine as hepatotoxins.TABLE 4 Hepatoprotective activity (in vivo) of synthetic TCA vis-a-visnatural fed at 48 h, 24 h, 2 h before and 6 h after CCl₄ (0.5 ml. kg⁻¹,p.o.) induced hepatic injury in rats^(a). NATURAL SYNTHETIC SILYMARINTCA 50 mg. TCA 50 mg. 50 mg. kg⁻¹, p.o. kg⁻¹, p.o. kg⁻¹, p.o. PARAMETERSPercent Hepatoprotection S GPT 67.22 74.63 59.73 S GOT 59.38 62.16 53.89S ALP 72.15 81.78 67.87 S BILIRUBIN 62.22 55.55 51.11 S TRIGLYCERIDES63.77 66.05 41.91 HEPATIC LP 61.72 64.33 77.58 HEPATIC GSH 59.06 52.9261.98

[0147] TABLE 3 Hepatoprotective activity (in vivo) of TCA (Synthetic)fed at 48 h, 24 h, 2 h before and 6 h after CCl₄(0.5 ml.kg⁻¹, p.o.)induced hepatic injury in rats^(a). Dose mg/ Serum parameters Hepaticparameters kg GPT GOT Bilirubin Triglycerides Lipid Treatment p.o.(Units) (Units) ALP^(b) (mg %) (mg %) peroxidation^(c) Glutathione^(d)Vehicle —  108.44 ±  119.12 ± 14.92  32.51 ± 1.94   0.13 ± 0.02  11.66 ±0.40  43.13 ± 3.16   5.21 ± 0.28 Control  16.05 Vehicle + — 1821.52 ±1062.29 ± 58.28  88.23 ± 6.60   0.72 ± 0.03  46.08 ± 3.84  89.38 ± 7.51  2.86 ± 0.43 CCl₄  126.46 TCA 100  87.25 ±  103.37 ± 18.66  27.25 ±1.72   0.18 ± 0.02   9.02 ± 0.84  40.41 ± 2.84   6.02 ± 0.35 Alone 23.80 TCA + 12.5  113.95 ±  798.85 ± 45.04**  67.10 ± 4.28**   0.56 ±0.02**  40.38 ± 2.86^(NS)  73.33 ± 6.64^(NS)   3.61 ± 0.37^(NS) CCl₄ 76.67**   (39.84 ±   (27.93 ± 4.77) (38.01 ± 7.72) (26.60) (23.14 ±4.13) (34.70 ± 14.35) (38.79 ± 12.39)   4.47) TCA + 25  973.56 ±  623.73± 57.95**  61.53 ± 3.35**   0.47 ± 0.02**  34.48 ± 3.29*  66.09 ± 3.56*  3.87 ± 0.21^(NS) CCl₄  76.45**   (49.49 ±   (46.49 ± 6.14)  47.92 ±5.94) (42.90 ± 3.83) (33.70 ± 9.58) (50.34 ± 7.71) (42.98 ± 8.70)  4.46) TCA + 50  829.44 ±  565.31 ± 60.33**  53.85 ± 3.49**   0.43 ±0.02**  28.03 ± 2.87**  59.60 ± 6.06**   4.13 ± 0.41^(NS) CCl₄  87.07**  (57.91 ±   (52.69 ± 6.39) (61.70 ± 6.22) (49.15 ± 3.96) (52.45 ± 8.35)(64.39 ± 13.11) (58.44 ± 14.85)   5.08) TCA + 100  690.23 ±  445.55 ±39.39**  48.96 ± 4.09**   0.36 ± 0.02**  22.77 ± 1.60**  48.69 ± 4.37**  4.50 ± 0.30** CCl₄  61.25**   (66.04 ±   (65.39 ± 4.18) (70.47 ± 7.35)(61.02 ± 4.13) (67.71 ± 3.37) (87.94 ± 9.44) (69.92 ± 12.77)   3.58)Silymarin + 50  840.86 ±  663.11 ± 49.03**  56.00 ± 2.85**   0.41 ±0.02**  32.26 ± 2.39**  62.73 ± 5.39**   4.46 ± 0.27* CCl₄  57.99**  (57.25 ±   (42.32 ± 5.19 (57.84 ± 5.11)  51.40 ± 4.77 (40.15 ± 6.94)(57.62 ± 11.66) (68.01 ± 11.90)   3.39)

[0148] TABLE 5 Hepatoprotective activity (in vivo) of ICA (Natural) fedat 6 h, 24 h, 48 h after CCl₄ (0.5 ml. kg−¹, p.o.) induced hepaticinjury in rats^(a). Dose Serum parameters mg/kg GPT GOT BilirubinTriglycerides Treatment p.o. (Units) (Units) ALP^(b) (mg %) (mg %)Vehicle Control —  91.50 ± 12.53  97.22 ± 7.75  23.50 ± 2.58   0.12 ±0.02   8.11 ± 0.65 Vehicle + CCl₄ — 1512.97 ± 72.25 776.34 ± 80.61 65.07 ± 6.11   0.88 ± 0.06  14.08 ± 1.00 TCA Alone 100  92.18 ± 15.75 82.13 ± 10.59  25.84 ± 2.32   0.16 ± 0.02   6.98 ± 0.86 TCA + CCl₄ 12.51001.86 ± 48.55** 524.23 ± 34.93**  51.81 ± 3.94^(NS)   0.54 ± 0.02** 12.57 ± 0.61^(NS)   (35.59 ± 3.37)  (37.12 ± 5.14) (27.87 ± 9.48)(44.52 ± 3.66) (24.12 ± 10.07) TCA + CCl₄ 25  772.68 ± 47.66** 448.28 ±54.88**  50.92 ± 1.62*   0.47 ± 0.03**  11.81 ± 1.05^(NS)   (51.52 ±3.31)  (48.31 ± 8.808) (34.03 ± 3.89) (53.51 ± 4.06) (36.23 ± 18.23)TCA + CCl₄ 50  582.12 ± 37.7** 365.43 ± 29.43**  43.63 ± 2.82**   0.39 ±0.02**  11.06 ± 0.61*   (64.76 ± 2.62)  (60.50 ± 4.33) (51.57 ± 6.78)(64.25 ± 3.76) (48.86 ± 10.64) TCA + CCl₄ 100  457.55 ± 18.18** 318.96 ±16.15**  32.93 ± 2.13**   0.29 ± 0.02**  10.37 ± 0.65**   (73.41 ± 1.26) (67.35 ± 2.38) (77.31 ± 5.13) (76.54 ± 3.66) (61.02 ± 11.24)Silymarin + CCl₄ 50  677.55 ± 53.72** 413.03 ± 24.98**  48.44 ± 2.98**  0.46 ± 0.03**  11.04 ± 0.54^(NS)   (58.16 ± 3.74)  (53.49 ± 3.67)(39.99 ± 7.17) (55.26 ± 4.05) (42.48 ± 8.75)

[0149] TABLE 6 Hepatoprotective activity (in vivo) of TCA (Natural) fedat 72 h, 48 h, 24 h, 1 h before inhalation of diethyl-ether and 1 hafter acetaminophen (200. mg.kg⁻¹, i. p., 6 h after exposure todiethyl-ether) in mice^(a). Dose Serum parameters mg/kg, GPT GOTTriglycerides Treatment p.o. (Units) (Units) ALP^(b) (mg %) VehicleControl —  179.47 ± 30.85  129.74 ± 27.28  25.06 ± 2.65   8.76 ± 0.93Vehicle + APAP — 2775.87 ± 138.60 1407.19 ± 51.34  57.21 ± 2.24  17.07 ±1.33 TCA Alone 100  146.00 ± 32.15  106.86 ± 30.15  27.15 ± 2.92   7.77± 0.56 TCA + APAP 12.5 2182.19 ± 137.86 1079.43 ± 98.66  50.41 ± 3.07 14.72 ± 0.68   (22.86 ± 5.31)   (25.66 ± 7.72) (21.27 ± 9.50) (28.28 ±8.27) TCA + APAP 25 1696.53 ± 48.31  880.10 ± 60.29  46.23 ± 1.73  13.47± 0.89   (41.57 ± 1.86)   (41.26 ± 4.72 (34.15 ± 5.39) (43.32 ± 10.80)TCA + APAP 50 1066.99 ± 118.14  619.36 ± 88.89  41.44 ± 1.27  12.34 ±0.85   (65.82 ± 4.55)   (61.67 ± 6.96) (49.04 ± 3.94) (56.84 ± 10.23)TCA + APAP 100  539.60 ± 51.65  310.53 ± 45.32  35.08 ± 2.18  10.87 ±0.69   (86.13 ± 1.99)   (85.85 ± 4.25) (68.82 ± 6.78) (74.48 ± 8.37)Silymarin + APAP 50 1060.09 ± 88.87  716.83 ± 44.44  42.53 ± 1.90  12.48± 0.86   (65.97 ± 3.44)   (53.52 ± 2.17) (45.66 ± 5.93) (55.27 ± 10.39)

[0150] TABLE 2 Hepatoprotective activity (in vivo) of TCA (Natural) fedat 48 h, 24 h, 2 h before and 6 h after CCl₄ (0.5 ml. kg⁻¹, p.o.)induced hepatic injury in rats^(a). Dose Serum parameters mg/kg GPT GOTBilirubin Triglycerides Treatment p.o. (Units) (Units) ALP^(b) (mg %)(mg %) Vehicle Control —  130.91 ± 3017.33 138.12 ± 32.21  21.60 ± 2.16  0.13 ± 300.02   8.71 ± 1.06 Vehicle + CCl₄ — 1604.01 ± 100.32 980.50 ±37.95  53.13 ± 3.56   0.79 ± 0.03  15.23 ± 1.58 TCA Alone 100  102.66 ±28.41 110.53 ± 25.55  23.15 ± 2.92   0.17 ± 0.02   6.98 ± 0.79 TCA +CCl₄ 12.5  992.34 ± 73.25** 695.16 ± 67.28**  45.25 ± 2.37^(NS)   0.47 ±0.O1**  13.51 ± 0.51^(NS)   (41.52 ± 4.97)  (33.87 ± 7.99) (26.32 ±6.55) (47.98 ± 2.02) (26.40 ± 7.84) TCA + CCl₄ 25  924.39 ± 39.82**674.44 ± 53.20**  39.48 ± 2.46**   0.42 ± 0.01**  12.11 ± 070^(NS)  (46.10 ± 2.70)  (36.33 ± 6.32) (43.28 ± 7.81) (55.33 ± 2.86) (47.72 ±11.33) TCA + CCl₄ 50  636.69 ± 44.49** 543.99 ± 79.93**  35.19 ± 2.90**  0.34 ± 0.01**  11.89 ± 0.75^(NS)   (65.66 ± 3.02)  (51.82 ± 9.94)(56.81 ± 9.15) (67.67 ± 2.30) (51.77 ± 11.46) TCA + CCl₄ 100  419.72 ±17.60** 432.08 ± 37.88**  30.14 ± 2.54**   0.26 ± 0.02**  11.20 ±0.68^(NS)   (80.39 ± 1.19)  (65.10 ± 4.50) (72.92 ± 8.07) (79.04 ± 3.57)(61.75 ± 1.44) Silymarin + 50  809.18 ± 45.35** 543.60 ± 45.91**  36.24± 1.77**   0.40 ± 0.03**  12.07 ± 0.97^(NS) CCl₄   (54.04 ± 2.99) (50.68 ± 6.36) (53.56 ± 5.63) (59.09 ± 5.44) (48.46 ± 14.96)

[0151] TABLE 1 Hexobarbitone sleep time and zoxazolamine paralysis time(in vivo) of TCA (Natural) fed at 1 h before CCl₄ (50 μl. kg⁻¹, p.o.)administration in mice^(a) Dose Hexobarbitone Zoxazolamine Treatment mgkg⁻¹ p.o. Sleeping time (min) Hepatoprotection (%) Paralysis time (min)Hepatoprotection (%) Vehicle Control — 23.33 ± 1.76 — 21.33 ± 1.36 —Vehicle + CCl₄ — 55.66 ± 1.87**b — 50.83 ± 3.10**^(b) TCA Alone 10021.85 ± 1.34 — 19.94 ± 1.49 — TCA + CCl₄ 12.5 44.16 ± 1.87*c 35.55 ±5.78 39.00 ± 1.93*c 40.10 ± 6.55 TCA + CCl₄ 25 37.83 ± 2.31*c 55.14 ±7.16 35.66 ± 3.28**c 51.41 ± 11.12 TCA + CCl₄ 50 31.66 ± 2.31**c 74.21 ±6.27 30.00 ± 2.26**c 70.61 ± 7.68 TCA + CCl₄ 100 28.16 ± 2.30**c 85.04 ±7.12 26.00 ± l.93**c 84.17 ± 6.55 Silymarin + CCl₄ 50 36.33 ± 2.46**c59.78 ± 7.60 34.66 ± 1.68**c 54.79 ± 5.72

[0152] TABLE 7 Hepatoprotective activity (in vivo) of TCA (Natural) fedat 1 h, 24 h, 48 h, 72 h after acetaminophen (200. mg. kg⁻¹, i. p. 6 hafter exposure to diethyl-ether) in mice^(a). Dose Serum parametersmg/kg, GPT GOT Triglycerides Treatment p.o. (Units) (Units) ALP^(b) (mg%) Vehicle Control —  145.94 ± 21.48  129.74 ± 27.28  25.06 ± 2.65  8.76 ± 0.93 Vehicle + APAP —  682.11 ± 83.73 1407.19 ± 51.34  57.21 ±2.24  17.07 ± 1.33 TCA Alone 100  129.65 ± 24.84  133.77 ± 20.84  22.57± 2.95   7.43 ± 0.89 TCA + APAP 12.5  431.85 ± 25.52** 1079.43 ±98.66^(NS)  50.41 ± 3.07^(NS)  14.72 ± 0.68^(NS)   (49.67 ± 2.81)  (25.66 ± 7.72) (21.27 ± 9.50) (28.28 ± 8.27) TCA + APAP 25  320.07 ±41.33**  880.10 ± 60.29*  46.23 ± 1.73*  13.47 ± 0.89^(NS)   (67.52 ±7.71)   (41.26 ± 4.72) (34.15 ± 5.39) (43.32 ± 10.80) TCA + APAP 50 282.74 ± 32.96**  619.36 ± 88.89**  41.44 ± 1.27**  12.34 ± 0.85**  (74.48 ± 6.154.55)   (61.67 ± 6.96) (49.04 ± 3.94) (56.84 ± 10.23)TCA + APAP 100  235.89 ± 22.7**  310.53 ± 45.32**  35.08 ± 2.18**  10.87± 0.69**   (83.33 ± 1.99)   (85.85 ± 4.25) (68.82 ± 6.78) (74.48 ± 8.37)Silymarin + APAP 50 1060.09 ± 88.87**  348.64 ± 344.76**  42.53 ± 1.90* 12.48 ± 0.86*   (65.97 ± 3.44)   (55.22 ± 8.39) (45.66 ± 5.93) (55.27 ±10.39)

[0153] TABLE 9 Hepatoprotective activity (in vivo) of TCA (Natural) fedat 6 h, 24 h, 48 h and 72 h after GalN (300. mg. kg⁻¹, s.c.) inducedhepatic injury in rats^(a). Dose Serum parameters mg kg⁻¹, GPT GOTBilirubin Triglycerides Treatment p.o. (Units) (Units) (mg %) ALP^(b)(mg %) Vehicle Control —  95.23 ± 7.82  87.37 ± 6.88   0.15 ± 0.02 26.99 ± 2.53   5.79 ± 0.93 Vehicle + GalN — 905.48 ± 58.27 705.43 ±49.39   0.92 ± 0.03  60.78 ± 3.72  14.81 ± 2.54 TCA Alone 50 105.02 ±18.74  91.07 ± 10.52   0.19 ± 0.02  24.45 ± 2.76   6.44 ± 0.38 TCA +GalN 12.5 578.14 ± 57.37** 494.53 ± 41.13**   0.56 ± 0.03**  46.03 ±2.35^(NS)  11.85 ± 0.75^(NS)  (40.40 ± 7.08)  (34.12 ± 6.65) (46.75 ±3.74) (43.65 ± 6.96) (32.78 ± 8.37) TCA + GalN 25 518.66 ± 42.64**449.93 ± 31.58**   0.46 ± 0.03**  38.87 ± 2.62**  10.48 ± 0.91^(NS) (47.74 ± 5.26)  (41.37 ± 5.10) (59.74 ± 4.04) (64.83 ± 7.74) (46.11 ±9.77) TCA + GalN 50 412.17 ± 31.17** 391.66 ± 29.53**   0.35 ± 0.02** 36.94 ± 1.87**   9.23 ± 059*  (60.88 ± 3.85)  (50.76 ± 4.78) (73.16 ±2.95) (71.88 ± 5.54) (61.84 ± 6.59) Silymarin + GalN 50 475.43 ± 53.47**393.83 ± 26.41**   0.45 ± 0.03**  45.24 ± 3.01^(NS)  12.39 ± 1.58^(NS) (53.07 ± 6.60)  (50.41 ± 4.27) (60.17 ± 4.17) (45.99 ± 8.91) (26.75 ±17.58)

[0154] TABLE 8 Hepatoprotective activity (in vivo) of TCA (Natural) fedat 72 h, 48 h, 24 h, 2 h before and 6 h after GalN 300. mg. kg⁻¹, s.c.)induced hepatic injury in rats^(a). Dose Serum parameters mg kg⁻¹, GPTGOT Bilirubin Triglycerides Treatment p.o. (Units) (Units) (mg %)ALP^(b) (mg %) Vehicle Control —  97.98 ± 9.97 116.94 ± 20.76   0.17 ±0.02  22.92 ± 1.89   7.48 ± 0.85 Vehicle + GalN — 1161.74 ± 54.83 859.24± 60.38   0.82 ± 0.03  59.26 ± 2.63  37.84 ± 2.58 TCA Alone 50  95.22 ±8.74 122.47 ± 11.84   0.20 ± 0.02  19.84 ± 1.58   8.79 ± 0.55 TCA + GalN12.5  743.62 ± 66.83** 613.20 ± 65.19**   0.56 ± 0.03**  47.07 ± 2.98** 26.49 ± 1.88*   (39.30 ± 6.25)  (34.60 ± 7.57) (39.49 ± 4.59) (30.88 ±7.77) (37.38 ± 6.18) TCA + GalN 25  635.11 ± 54.67** 505.26 ± 29.63**  0.44 ± 0.05**  40.49 ± 2.29**  23.58 ± 2.02**   (49.35 ± 5.16)  (47.68± 3.99) (58.00 ± 3.86) (51.62 ± 6.31) (46.97 ± 6.67) TCA + GalN 50 413.53 ± 23.94** 406.05 ± 25.82**   0.36 ± 0.02**  34.94 ± 1.43** 21.37 ± 1.51   (70.34 ± 2.25)  (61.05 ± 3.48) (70.76 ± 2.78) (66.91 ±3.92) (54.25 ± 4.97) Silymarin + GalN 50  587.98 ± 54.59** 458.13 ±40.69**   0.44 ± 0.02**  42.08 ± 2.56**  28.85 ± 1.85*   (53.94 ± 5.13) (54.03 ± 5.48) (58.71 ± 4.13) (47.25 ± 7.05) (29.61 ± 6.08)

[0155] TABLE 10 Hepatoprotective activity (in vivo) of TCA (Natural)against alcohol induced hepatic damage in rats^(a). Gr.III Gr. IV Gr.VSerum Gr.I Gr.II TCA (25 mgkg⁻¹) + TCA (50 mgkg⁻¹) + Silymarin (50 mgkg⁻¹) + Parameters Units Vehicle Control Vehicle + Alcohol AlcoholAlocohol Alcohol SGOT U/l 296.00 ± 9.79 357.00 ± 33.39 310.00 ±30.36^(*b) 295.00 ± 34.39^(*b) 333.00 ± 29.71^(**b) % protection — —77.29 101.09  39.43 SGPT U/l 199.00 ± 16.34 281.00 ± 12.39 234.00 ±8.80^(**b) 220.00 ± 11.30^(*b) 224.00 ± 11.76^(**b) % protection — —57.33  73.86  49.03 GGT IU/l  1.42 ± 0.303  2.69 ± 0.701  1.90 ± 0.447 1.54 ± 0.128  1.43 ± 0.157 % protection — — 63.00  90.00 100.00 ALP U/l 14.00 ± 1.23  24.00 ± 1.60  16.00 ± 2.04^(***b)  14.60 ± 2.82^(***b) 13.80 ± 2.016^(**b) % protection — — 80.00  95.00 100.00 Protein g/dl 11.70 ± 3.14  10.50 ± 2.10  16.90 ± 3.78  12.60 ± 3.09  10.80 ± 3.26 %protection — − 10.30 44.40  7.70  −7.70

[0156] TABLE 11 Hepatoprotecive activity (in vivo) of RLJ-NE-598(025)(F)(A₃)(N) against alcohol induced hepatic damage in rats^(a). Gr.III Gr. IV Gr. V Gr. II (F)(A₃)(N) (F)(A₃)(N) Silymarin Hepatic Gr IVehicle + (25 mgkg⁻¹) + (50 mgkg⁻¹) (50 mg kg⁻¹) + Parameters UnitsVehicle Control alcohol Alcohol Alcohol Alcohol SDH Units/g  9.13 ±0.196  7.58 ± 0.338**^(b)  7.79 ± 0.272  8.24 ± 0.390  8.18 ± 0.113^(*c)% protection — — 14 43 39 G-6-Pase Units/g  2.36 ± 0.235  1.73 ± 0.135 2.19 ± 0.465  2.20 ± 0.223  2.20 ± 0.356 % protection — — 73 75 75Protein mg/g 179.00 ± 8.23   168 ± 7.51   172 ± 20.430   174 ± 2.11  181 ± 6.89 % Change — −7.1 −3.9 −2.8  1.1 TG mg/g  15.22 ± 4.874 16.56± 1.496 14.75 ± 0.60 15.44 ± 1.073 14.67 ± 2.589 % Change —  8.8 −3.1 1.4 −3.6

[0157] TABLE 12 Cholretic activity of TCA and dehydrocholic acid inrats^(a). Bile parameters^(a) % Increase (as compared to normal) DoseBile flow Bile solids Treatment mg. kg⁻¹ Route (mg %) (mg %) TCA 50 i.d. 8.23 ± 1.71 13.40 ± 1.53 TCA 20 i.v. 43.63 ± 3.13 31.36 ± 4.09 TCA 50i.d.  38.4 ± 2.76 28.13 ± 3.89

1. A pharmaceutical composition comprising an effective amount ofbioactive trans-tetracos-15-enoic acid extracted from a plant Indigoferatinctoria, used for the treatment of subjects with hepatic disorders. 2.A pharmaceutical composition according to claim 1 wherein saidcomposition along with dehydrocholic acid is used for the enhancement ofcholretic activity.
 3. A composition according to claim 1 whereintrans-tetracos-15-enoic acid is used singly or in combination withpharmaceutically acceptable additives.
 4. A composition according toclaim 1 wherein the pharmaceutically acceptable additives are selectedfrom the group consisting of carriers, diluents, solvents, filterslubricants, excipients, binder or stabilizers.
 5. A compositionaccording to claim 1 wherein the said composition is used for bothpreventive and curative properties.
 6. A composition according to claim1 wherein said composition is used systemically, orally or by anyclinically/medically accepted methods.
 7. A composition according toclaim 1 wherein the composition is used to treat hepatic disorders thatare clinically, biochemically and histologically similar to that ofviral hepatitis, chronic hepatitis, fatty liver, cirrhosis and severalvascular lesions of the liver.
 8. A composition according to claim 1wherein said composition is used to treat damage induced byhepatotoxins.
 9. A composition according to claim 1 wherein thehepatotoxins are selected from the group comprising Galactosamine,Paracetamol, Carbon tetrachloride and alcohol.
 10. A compositionaccording to claim 1, wherein the subjects is selected from the groupconsisting of humans and mammals, preferably humans.
 11. A compositionaccording to claim 1 wherein the dosage for the treatment of CCl₄induced hepatotoxicity in mammals is in the range of 10-100 mg/kg bodyweight.
 12. A composition according to claim 1 wherein the preferreddosage for the treatment of CCl₄ induced hepatotoxicity in mammals is inthe range of 50-100 mg/kg body weight.
 13. A composition according toclaim 1 wherein the hepatoprotective activity in CCl₄ inducedhepatotoxic mammals is upto 88%.
 14. A composition according to claim 1wherein the dosage for the treatment of acetaminophen inducedhepatotoxicity in mammals is in the range of 10-100 mg/kg body weight.15. A composition according to claim 1 wherein the preferred dosage forthe treatment of acetaminophen induced hepatotoxicity in mammals is inthe range of 50-100 mg/kg of body weight.
 16. A composition according toclaim 1 wherein the hepatoprotective activity in acetaminophen inducedhepatotoxicity in mammals is upto 86%.
 17. A composition according toclaim 1 wherein the dosage for the treatment of Galactosamine inducedhepatotoxicity in mammals is in the range of 10-50 mg/kg of body weight.18. A composition according to claim 1 wherein the preferred dosage forthe treatment of D-Galactosamine induced hepatotoxicity is in the rangeof 25-50 mg/kg of body weight.
 19. A composition according to claim 1wherein the hepatoprotective activity in Galactosamine inducedhepatotoxicity in mammals is upto 73%.
 20. A composition according toclaim 1 wherein the dosage for the treatment of alcohol inducedhepatotoxicity in mammals is in the range of 10-50 mg/kg of body weight.21. A composition according to claim 1 wherein the preferred dosage forthe treatment of alcohol induced hepatotoxicity is in the range of 25-50mg/kg of body weight.
 22. A composition according to claim 1 wherein thehepatoprotective activity in alcohol induced hepatotoxicity in mammalsis upto 100%.
 23. A composition according to claim 1 wherein thepreferred dosage for hepatic disorders in mammals is about 50-100 mg/kgof body weight.
 24. A composition according to claim 1 wherein thepreferred dosage for hepatic disorders in human beings is about 10-15mg/kg of body weight.
 25. A process for the isolation oftrans-tetracos-15-enoic acid, a bioactive constituent from the plantIndigofera tinctoria, said process comprising the steps of: (a)extracting the powdered plant parts with an aliphatic hydrocarbonsolvent at a temperature in the range of 60-80° C. for about 20-30hours; (b) triturating the extract of step (a) with a ketonic solvent;(c) separating the solvent soluble portion of step (b) and the residueby using silica gel bed; (d) subjecting the residue of step (c) tocolumn chromatography by eluting with a mixture of organic solvents ofincreasing polarity to yield a bioactive fraction; and (e) resolvingfraction as obtained in step (e) by high performance liquidchromatography and crystallizing with an alcoholic solvent to obtain thebio-active constituent trans-tetracos-15-enoic acid.
 26. A processaccording to claim 24 wherein alternatively, the fraction obtained instep (d) when subjected to semipreparative high performance liquidchromatography, yields bioactive constituent trans-tetracos-15-enoicacid.
 27. A process according to claim 25 wherein the plant parts usedare aerial parts selected from the group consisting of leaves, stem andbark.
 28. A process according to claim 25 wherein the hydrocarbonsolvent is selected from the group consisting of petroleum ethane,n-hexane, cyclohexane or ligroin and preferably petroleum ether.
 29. Aprocess according to claim 25 wherein in the ketonic solvent is selectedfrom the group consisting of acetone, ethyl-methyl-ketone, or methylisobutyl ketone.
 30. A process according to claim 25 wherein thesolvents used for eluting the column to obtain hepatoprotective fractionare selected from the group consisting of petroleum ether, n-hexane,chloroform, ethyl acetate, a mixture thereof and preferably a mixture ofpetroleum ether and ethyl acetate.
 31. A process according to claim 25wherein in step (d), the solvent used for performing HPLC is a mixtureof Acetonitrile and H₂O in the ratio of 95:5.
 32. A process as accordingto claim 25 wherein in step (e) the HPLC purified compound is furtherpurified by crystallizing with an alcoholic solvent selected frommethanol and isopropanol.
 33. A process according to claim 25 whereinthe hepatoprotective fraction thus obtained is designated as Indigotoneconsisting of a mixture of three compounds.
 34. A process according toclaim 25 wherein the percentage of mixture of three compounds ofhepatoprotective fraction is 11-12%, 12-13% and 75-77% respectively. 35.A process according to claim 25 wherein the hepatoprotective constituentobtained by the process of the present invention is to the extent of0.09-0.11% on the basis of dried plant material.
 36. A method oftreating subjects with liver disorders, the said method comprisingadministering a pharmaceutically effective dosage oftrans-tetracos-15-enoic acid.
 37. A method according to claim 36 whereintrans-tetracos-15-enoic acid is used to treat liver disorders caused byGalactosamine, Paracetamol, Carbon tetrachloride and alcohol.
 38. Amethod according to claim 36 wherein the dosage for the treatment ofCCl₄ induced hepatotoxicity in mammals is in the range of 10-100 mg/kgbody weight.
 39. A method according to claim 36 wherein the preferreddosage for the treatment of CCl₄ induced hepatotoxicity in mammals is inthe range of 50-100 mg/kg body weight.
 40. A method according to claim36 wherein the hepatoprotective activity in CCl₄ induced hepatotoxicmammals is upto 88%.
 41. A method according to claim 36 wherein thedosage for the treatment of acetaminophen induced hepatotoxicity inmammals is in the range of 10-100 mg/kg body weight.
 42. A methodaccording to claim 36 wherein the preferred dosage for the treatment ofacetaminophen induced hepatotoxicity in mammals is in the range of50-100 mg/kg of body weight.
 43. A method according to claim 36 whereinthe hepatoprotective activity in acetaminophen induced hepatotoxicity inmammals is upto 86%.
 44. A method according to claim 36 wherein thedosage for the treatment of Galactosamine induced hepatotoxicity inmammals is in the range of 10-50 mg/kg of body weight.
 45. A methodaccording to claim 36 wherein the preferred dosage for the treatment ofD-Galactosamine induced hepatotoxicity is in the range of 25-50 mg/kg ofbody weight.
 46. A method according to claim 36 wherein thehepatoprotective activity in Galactosamine induced hepatotoxicity inmammals is upto 73%.
 47. A method according to claim 36 wherein thedosage for the treatment of alcohol induced hepatotoxicity in mammals isin the range of 10-50 mg/kg of body weight.
 48. A method according toclaim 36 wherein the preferred dosage for the treatment of alcoholinduced hepatotoxicity is in the range of 25-50 mg/kg of body weight.49. A method according to claim 36 wherein the hepatoprotective activityin alcohol induced hepatotoxicity in mammals is upto 100%.
 50. A methodaccording to claim 36 wherein the dosage for the enhancement ofcholretic activity is in the range of 20-50 mg/kg of body weight.
 51. Amethod according to claim 36 wherein the enhanced cholretic activity inmammals is upto 43%.
 52. A method according to claim 36 whereintrans-tetracos-15-enoic acid is used singly or in combination withpharmaceutically acceptable carriers.
 53. A method according to claim 36wherein the trans-tetracos-15-enoic acid is administered to a subject incombination with pharmaceutically acceptable additives, carriers,diluents, solvents, filters, lubricants, excipients, binder orstabilizers.
 54. A method according to claim 36 wherein the desireddosage is administered for both preventive and curative properties. 55.A method according to claim 36 wherein trans-tetracos-15-enoic acid isadministered systemically, orally or by any clinically/medicallyaccepted methods.
 56. A method according to claim 36 wherein the subjectis selected from animals, mammals, and preferably humans.
 57. A methodaccording to claim 36 wherein the preferred dosage for hepatic disordersin mammals is 1 about 50-100 mg/kg of body weight.
 58. A methodaccording to claim 36 wherein the preferred dosage for hepatic disordersin human beings is about 10-15 mg/kg of body weight.